QIAxcel Advanced
fully
automates sensitive, high-resolution capillary
electrophoresis of up to 96 samples per run, streamlining your workflow
and reducing time to result. The new RNA Integrity Score (RIS) is an
objective quality measurement for eukaryotic RNA samples, which enables
fast and easy quality control of RNA integrity. QIAxcel takes ease of
use to a new level — just load the gel cartridge and buffer tray, place
your samples into the instrument, select the process profile, and go.
Say good-bye to tedious slab gel preparation and chip loading
procedures, and say hello to QIAxcel Advanced!

Generate comprehensive reports to view on any
computer or smart device

QIAxpert is an innovative high-speed microfluidic
UV/VIS spectrophotometer which profiles sample content to differentiate
between DNA and RNA and sample impurities. QIAxpert is fast and easy to
use, analyzing up to 16 samples within 2 minutes - just pipet
samples onto the QIAxpert slide, place it into the reader, and select
the pre-programmed method on the integrated touchscreen. QIAxpert is
the right tool for any fast-paced lab performing nucleic acid
quantification and quality control.

Bioanalyzer 2100 &
Experion

Microfluidics
deals with the behavior, precise control and manipulation of fluids
that are geometrically constrained to a small, typically sub-milimeter,
scale. It is a multidisciplinary field intersecting engineering,
physics, chemistry, microtechnology and biotechnology, with practical
applications to the design of systems in which such small volumes of
fluids will be used. Microfluidics has emerged in the beginning of the
1980s and is used in the development of inkjet printheads, DNA chips,
lab-on-a-chip technology, micro-propulsion, and micro-thermal
technologies.

It is generally known that total RNA quality has a
distinct influence on the validity and reliability of quantitative PCR
results. In addition, the recently published MIQE guidelines focus on
the pre-PCR steps and state the importance of RNA quality assessment.
Various studies showed the impairing effect of ongoing RNA degradation
on mRNA expression results. Therefore, the verification of RNA
integrity prior to downstream applications like RT-qPCR and
mircroarrays is indispensable. A fast and reliable assessment of RNA
integrity can be done with the Eukaryote Total RNA Nano Assay of the
Agilent 2100 Bioanalyzer. The importance of RNA quality should also be
considered in new applications such as the investigation of miRNA
expression profiles. With the Agilent Small RNA Assay, Agilent is
offering one of the few possibilities for selectively estimating miRNA
before expression analysis. However, by now little is known about
factors affecting miRNA analysis. Herein, the important impact of total
RNA quality on quantification of mRNA and miRNA should be considered.

Quantitative
real-time polymerase chain
reaction (qPCR) and microarray
analysis have become essential for elucidating variations in gene
expression. While guidelines that define the minimum information
required for interpretation of microarray data have been available
since 2001,[1] similar specifications for qPCR experiments have been
developed only recently. In early 2009, a consortium of leading
scientists who use qPCR, established specifications for the minimum
information that you must report for a qPCR experiment that you wish to
publish. These are the MIQE guidelines (for minimum information for
publication of quantitative real-time PCR experiments). This article
describes how the Agilent 2100 Bioanalyzer helps you meet these
requirements.

Purity and good
RNA quality are important elements for the overall
success of RNA based analysis methods like microarrays and real time
qRT-PCR. There are two commercially available automated systems – the
Experion (Bio-Rad Laboratories) and the 2100 Bioanalyzer (Agilent
Technologies) – that provide both RNA sample quality and quantity
analysis. In this study different aspects like the reproducibility and
sensitivity of both systems were analyzed by determining the total RNA
quality and quantity extracted from various bovine tissues. Regarding
quantitation, the Experion is more sensitive than the 2100 Bioanalyzer.
Both systems overstate the concentration by 19-29% compared to the
photometric values. For RNA quality determination, both systems show
highly comparable reproducibility. With the RNA integrity number (RIN)
the 2100 Bioanalyzer offers an additional opportunity to quantify the
RNA quality.

Successful
molecular analyses
of human solid tissues require intact biological material with
well-preserved nucleic acids, proteins, and other cell structures.
Pre-analytical handling, comprising of the collection of material at
the operating theatre, is among the first critical steps that influence
sample quality. The aim of this study was to compare the experimental
outcomes obtained from samples collected and stored by the conventional
means of snap freezing and by PAXgene Tissue System (Qiagen). These
approaches were evaluated by measuring rRNA and mRNA integrity of the
samples (RNA Quality Indicator and Differential Amplification Method)
and by gene expression profiling. The collection procedures of the
biological material were implemented in two hospitals during colon
cancer surgery in order to identify the impact of the collection method
on the experimental outcome. Our study shows that the pre-analytical
sample handling has a significant effect on the quality of RNA and on
the variability of qPCR data. PAXgene collection mode proved to be more
easily implemented in the operating room and moreover the quality of
RNA obtained from human colon tissues by this method is superior to the
one obtained by snap freezing.

Development
and Validation of RQ -- An RNA Quality Indicator for the Experion
Automated Electrophoresis Systemby Bio-Rad -- Bulletin
5761

The AdvanCE™ FS
Nucleic Acids Analyzer is a fluorescence-based capillary
electrophoresis instrument for both sizing and quantifying nucleic
acids (DNA and RNA). By using a sensitive intercalating dye coupled
with a powerful LED light source, AdvanCE™ FS Nucleic Acids Analyzer
obviates the need for fluorescent labeled primers and can be used to
separate dsDNA fragments and RNA (mRNA and total RNA). The AdvanCE™ FS
Nucleic Acids Analyzer is the most flexible system on the market today,
with the greatest sensitivity, the highest separation resolution over a
wide range, the widest dynamic range and the fastest separation times.

This instrument
comes available for customers who wish to run either 12 samples or 96
samples at a time.

Gels formulated to
provide high resolution separation are also available for general
fragment analysis, such as SSR/microsatellites, small and large PCR
fragments (amplicons) and resolve fragments sized between 10 bp and
40,000 bp with resolution down to 2bp.

Assessment of RNA
quality and RNA quantitation is critical for
many molecular biology techniques. Manual assessment methods for
both
quantity and quality can use large amounts of precious materials and
take a considerable amount of time to complete.

Several analytical
instruments exist for the evaluation of RNA.
However, these instruments either lack acceptable detection sensitivity
or require significant hands-on time to complete chip loading and
preparation. The AdvanCE FS process for RNA – either for total RNA
and/or messenger RNA – is completely automated, requiring minimal hands
on time.

The assessment of RNA
integrity is a critical ﬁrst step in obtaining meaningful gene
expression data. Working with
low-quality RNA may strongly compromise the experimental results of
downstream
applications which are often labour-intensive, time-consuming, and
highly expensive.
Using intact RNA is a key element for the successful application of
modern molecular biological methods,
like qRT-PCR or micro-array analysis. To verify RNA quality nowadays
commercially
available automated capillary-electrophoresis systems are available
which are
on the way to become the standard in RNA quality assessment. Proﬁles
generated yield information on RNA
concentration, allow a visual inspection of RNA integrity, and generate
approximated
ratios between the mass of ribosomal sub-units. In this review, the importance of RNA quality for
the qRT-PCR was analyzed by determining the RNA quality of diﬀerent
bovine tissues and
cell culture. Independent analysis systems are described and compared
(OD measurement,
NanoDrop, Bioanalyzer 2100 and Experion). Advantage and disadvantages
of RNA
quantity and quality assessment are shown in performed applications of
various tissues and cell cultures.
Further the comparison and correlation between the total RNA integrity
on PCR
performance as well as on PCR eﬃciency is described. On the basis of
the derived results we can argue that
qRT-PCR performance is aﬀected by the RNA integrity and PCR eﬃciency in
general is not
aﬀected by the RNA integrity.We can recommend a RIN higher than ﬁve as good total RNA
quality and
higher than eight as perfect total RNA for downstream application.Our
RNA integrity basis paper has been
frequently cited by other researchers:

mRNA and microRNA quality control for
RT-qPCR analysis
Becker C, Hammerle-Fickinger A, Riedmaier I, Pfaffl MW.
Physiology-Weihenstephan, Technical University Munich, Freising,
Germany.Methods. 2010 50(4): 237-243
The importance of high quality sample material, i.e. non-degraded or
fragmented RNA, for classical gene expression profiling is well
documented. Hence, the analysis of RNA quality is a valuable tool in
the preparation of methods like RT-qPCR and microarray analysis. For
verification of RNA integrity, today the use of automated capillary
electrophoresis is state of the art. Following the recently published
MIQE guidelines, these pre-PCR evaluations have to be clearly
documented in scientific publication to increase experimental
transparency. RNA quality control may also be integrated in the routine
analysis of new applications like the investigation of microRNA (miRNA)
expression, as there is little known yet about factors compromising the
miRNA analysis. Agilent Technologies is offering a new lab-on-chip
application for the 2100 Bioanalyzer making it possible to quantify
miRNA in absolute amounts [pg] and as a percentage of small RNA [%].
Recent results showed that this analysis method is strongly influenced
by total RNA integrity. Ongoing RNA degradation is accompanied by the
formation of small RNA fragments leading to an overestimation of miRNA
amount on the chip. Total RNA integrity is known to affect the
performance of RT-qPCR as well as the quantitative results in mRNA
expression profiling. The actual study identified a comparable effect
for miRNA gene expression profiling. Using a suitable normalization
method could partly reduce the impairing effect of total RNA integrity.mRNA and microRNA Purity and
Integrity: The Key to Success in Expression Profiling
Benedikt Kirchner, Vijay Paul, Irmgard Riedmaier and Michael W. PfafflChapter 5 -- in
Quantitative Real-Time PCR: Methods and Protocols (Methods
in Molecular Biology)
by Roberto Biassoni, Alessandro Raso

Relative quantification in quantitative real-time RT-PCR is
increasingly used to quantify gene expression changes. In general, two
different relative mRNA quantification models exist: the delta-delta Ct
and the efficiency-corrected Ct model. Both models have their
advantages and disadvantages in terms of simplification on the one hand
and efficiency correction on the other. The particular problem of RNA
integrity and its effect on relative quantification in qRT-PCR
performance was tested in different bovine tissues and cell lines (n =
11). Therefore different artificial and standardized RNA degradation
levels were used. Currently fully automated capillary electrophoresis
systems have become the new standard in RNA quality assessment. RNA
quality was rated according the RNA integrity number (RIN).
Furthermore, the effect of different length of amplified products and
RNA integrity on expression analyses was investigated. We found
significant impact of RNA integrity on relative expression results,
mainly on cycle threshold (Ct) values and a minor effect on PCR
efficiency. To minimize the interference of RNA integrity on relative
quantification models, we can recommend to normalize gene expression by
an internal reference gene and to perform an efficiency correction.
Results demonstrate that innovative new quantification methods and
normalization models can improve future mRNA quantification.Our
second RNA integrity basis paper has been
frequently cited by other researchers:http://Scholar.Google.com/Measurable impact of RNA quality on gene
expression results from quantitative PCR.
Vermeulen J, De Preter K, Lefever S, Nuytens J, De Vloed F, Derveaux S,
Hellemans J, Speleman F, Vandesompele J.
Nucleic Acids Res. 2011 39(9): e63

Compromised RNA quality is suggested to lead to unreliable results in
gene expression studies. Therefore, assessment of RNA integrity and
purity is deemed essential prior to including samples in the analytical
pipeline. This may be of particular importance when diagnostic,
prognostic or therapeutic conclusions depend on such analyses. In this
study, the comparative value of six RNA quality parameters was
determined using a large panel of 740 primary tumour samples for which
real-time quantitative PCR gene expression results were available. The
tested parameters comprise of microfluidic capillary electrophoresis
based 18S/28S rRNA ratio and RNA Quality Index value, HPRT1 5'-3'
difference in quantification cycle (Cq) and HPRT1 3' Cq value based on
a 5'/3' ratio mRNA integrity assay, the Cq value of expressed Alu
repeat sequences and a normalization factor based on the mean
expression level of four reference genes. Upon establishment of an
innovative analytical framework to assess impact of RNA quality, we
observed a measurable impact of RNA quality on the variation of the
reference genes, on the significance of differential expression of
prognostic marker genes between two cancer patient risk groups, and on
risk classification performance using a multigene signature. This study
forms the basis for further rational assessment of reverse
transcription quantitative PCR based results in relation to RNA quality.

The integrity of RNA is
a very critical aspect regarding downstream RNA based quantitative
analysis like RT-qPCR. Low-quality RNA can compromise the results of
such experiments. Today automated lab-on-chip capillary electrophoresis
allows rapid RNA quality and quantity determination, e.g. 2100
Bioanalyzer (Agilent Technologies) and the Experion (Bio-Rad). Both
platforms determine RNA quality using a numerical system which
represents the integrity of RNA. The Bioanalyzer offers the RIN
algorithm (RNA Integrity Number) on the Bioanalyzer 2100 and Bio-Rad
developed a new Experion software version that offers an algorithm for
calculating the RNA Quality Index (RQI).The aim of this study was to
compare both systems regarding sensitivity, reproducibility, linearity
and the influence of individual tissue extractions and different chip
runs on RNA quality and quantity determination.Overall it was confirmed
that both algorithms are very comparable and beneficial for the
determination of RNA quality for downstream applications. The Experion
showed slightly better results regarding reproducibility and absolute
sensitivity, whereas the 2100 Bioanalyzer showed a higher linearity.MIQE
GuidelinesRNA
Qualitätskontrolle in der Genexpressionsanalytik (in German)
Christiane Becker,
Irmgard Riedmaier, Michael W. Pfaffl
BIOspektrum - Special RNA Technologien 2009 (5): 512 - 515